Neurotransmission requires the fusion of synaptic vesicles with the plasma membrane, which releases neurotransmitter into the synaptic cleft. Proteins called soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate this fusion process by bringing the two membranes together and catalyzing fusion. Canonically, calcium entry during action potential stimulation of presynaptic terminals causes synchronous fusion of many synaptic vesicles arising from multiple synapses. In the absence of action potentials, however, some vesicles fuse spontaneously, releasing neurotransmitter in a sparse and temporally random pattern. It is unclear whether this spontaneous neurotransmission shapes neuronal function and alters synaptic plasticity and behavior, but prior work has implicated its inhibition in the fast-acting antidepressant effects of low-dose ketamine administration in treatment-resistant depression. Recently, two noncanonical vesicular SNAREs, vps10p tail interactor 1a (vti1a) and vesicle-associated membrane protein 7 (VAMP7/TI- VAMP), were identified as specific modulators of spontaneous neurotransmission. Reductions in these proteins in vitro reduce the frequency of spontaneous fusion events. The overall goal of the proposed project is to identify the roles spontaneous neurotransmission plays in vivo, especially in the fast-acting antidepressant response to ketamine. To achieve this goal, vti1a and VAMP7 will be knocked down in vivo via stereotaxic injection of virus encoding shRNA into the hippocampus, an area thought to be important for ketamine's effects. The first aim of the proposed project is to determine whether vti1a and VAMP7 knockdown alters spontaneous neurotransmission and synaptic plasticity by using a combination of electrophysiological and optical recording methods. The second aim of the proposed project is to determine whether knockdown of vti1a and VAMP7 modifies ketamine's ability to produce antidepressant responses. This will be tested by measuring behavior after ketamine treatment in mice that were previously injected with the virus knocking down vti1a and VAMP7. Together, these aims will determine whether a reduction in levels of vesicular SNAREs vti1a and VAMP7 alters synaptic function and in vivo behavior. Additionally, this project will clarify upstream mechanisms responsible for the fast-acting antidepressant effects of ketamine, potentially leading to identification of additional signaling cascades that can be modified to treat depression while avoiding the abuse potential inherent in ketamine administration. This proposed project also provides valuable training to the applicant in developing techniques and professional skills important for a career in biomedical research.